Project summary The goal of this proposal is to understand how heterochromatin can physically impede the binding of activator proteins and thus impede cellular reprogramming to pluripotency and other fate changes. Of all the forms of directed conversion of one cell fate to another, the conversion of somatic cells to pluripotency (iPS) is the most robust. Yet all forms of cell type conversion, including to iPS cells, remain inefficient, with a stochastic period that precedes a deterministic, developmental path to the terminal state. Various screens have identified genes whose impairment results in enhanced cell fate conversion, yet the underlying mechanisms are often obscure. We discovered a specific epigenetic impediment, H3K9me3- based heterochromatin, whose mechanism of action is by physically blocking the binding of reprogramming transcription factors. H3K9me3-heterochromatin blankets genes at the top of a developmental hierarchy, explaining how loss of H3K9me3 promotes cell conversion. We now find that such domains can explain deficiencies in direct cell reprogramming to other fates, demonstrating the generality of the mechanism. Dynamics in H3K9me3 heterochromatin are also involved in X chromosome inactivation, which we will investigate with Kathrin Plath. We developed methods to physically isolate H3K9me3-heterochromatin from somatic cells, determine its protein composition, and perform functional screens, thereby establishing our premise that heterochromatic proteins play selective roles in impairing the ability of H3K9me3-embedded genes to be induced by reprogramming transcription factors. Relevant proteins overlap with studies of Konrad Hochedlinger. The central theme guiding this proposal, based on our latest data, is that there are different heterochromatin complexes blocking different sets of protein coding genes, and that by understanding the composition and function of such complexes, we will change cell fate far more selectively and efficiently than by targeting all H3Kme3-based heterochromatin. We therefore propose: Aim 1. We will identify heterochromatin components and specific genomic complexes that impede the action of reprogramming transcription factors and, with K. Plath, that maintain an inactive X chromosome. Aim 2. We will define the function of heterochromatin components and subcomplexes, including those involved in RNA processing and others with K. Hochedlinger. Our mechanistic understanding will provide a more rational and selective ability to activate different types of heterochromatic genes, and thereby more specifically enhance cellular reprogramming for disease models and cell therapeutics in the future.